A key paradigm in urothelial biology is that urothelial umbrella cells can reversibly adjust its apical cell surface area. Thus bladder expansion can trigger the cytoplasmic fusiform vesicles to fuse with the apical surface, while bladder contraction can lead to the retrieval of some apical surface membranes to reform fusiform vesicles. Central to this paradigm is a group of urothelium-specific proteins, the uroplakins, that are made as major urothelial differentiation products. Uroplakins form 16-nm particles packed hexagonally to form 2D crystals of urothelial plaques that constitute almost the entire urothelial apical surface as well as the fusiform vesicles. We have previously demonstrated that uroplakins contribute to bladder barrier function, that uroplakin defects may lead to severe renal dysplasia, that cessation of uroplakin expression is associated with unfavorable bladder cancer outcome, and that uroplakin Ia is the urothelial receptor for uropathogenic E. coli. These findings establish not only the fundamental importance of uroplakins in bladder barrier function, but also their possible involvements in major urological diseases. Despite the potential importance of uroplakins in urothelial biology and diseases, little is known about how these proteins assemble into urothelial plaques, how they are delivered selectively to the apical cell surface, and how they are endocytosed/retrieved from the apical surface. Given the recent data suggesting that uropathogenic E. coli, after binding to uroplakin Ia receptor, invade into the umbrella cells via the uroplakin endocytic pathway, it is crucial that we better understand these fundamentally important urothelial cellular processes and their regulation. We are in an excellent position to study uroplakin trafficking because we have recently identified a major urothelial plaque-associated protein, MAL, that is likely to be involved in the apical delivery of uroplakins. Similarly, we have discovered a novel, urothelium-specific sorting nexin, tentatively named SNX32, that may be involved in uroplakin retrieval. Thus, the goals of our studies during the next funding period are to better understand: (i) how uroplakins assemble into a 2D crystalline plaque;(ii) how uroplakins are delivered to apical surface;and (iii) how uroplakins are retrieved for degradation and/or recycle. Our results should yield new insights into the mechanisms of bladder barrier function, reversible urothelial surface area adjustment, as well as urological diseases including urinary tract infection. PUBLIC HEALTH RELEVANCE: Urothelial plaques are unique structures covering the apical surface of urinary bladder urothelium, and play key roles in bladder barrier function and bacterial infection. Our proposed studies on how the urothelial plaques, that harbor the bacterial receptor, are assembled, delivered to the urothelial apical surface, and retrieved for degradation. These studies can have important implications for urinary tract infection, interstitial cystitis, urinary tract obstruction and renal adysplasia.